Method for inhibiting apoptosis under ischemic condition

ABSTRACT

The present invention relates to a method for inhibiting apoptosis under ischemic condition, which comprises a step of administering antibiotics of quinolones, quinones, aminoglycosides or chloramphenicol to an individual under ischemic condition which lacks an adequate supply of oxygen and glucose. In accordance with the present invention, the antibiotics increase cell viability under hypoxic and hypoglycemic condition, assuring that they can be applied as a therapeutic agent for ischemia-associated diseases such as myocardial infarction and cerebral infarction.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for inhibiting apoptosis, more specifically, to a method for inhibiting apoptosis under ischemic condition.

[0003] 2. Description of the Prior Art

[0004] As the death rate from cardiovascular diseases is increasing recently, researches on the cardiovascular diseases are now in rapid progress. Among them, one of the most noticeable field is that relating to thrombus, wherein efforts to restore blood vessel functions by dissolving thrombus which is the major cause of blockage of blood vessels, and, furthermore, to inhibit thrombus formation are being made. However, there is little progress in developing method for preventing ripple effects of blockage of blood vessels caused by thrombus or other causes. Accordingly, in case of a patient dying of the blockage of blood vessels, it is almost impossible to alleviate ischemic injury which lacks an adequate supply of oxygen and glucose.

[0005] It has been reported that administration of antibiotics to the patient who has antibody against Chlamydia pneumoniae which is related to onset of acute myocardial infarction reduces the onset rate of acute myocardial infarction (see: Meier C. R. et al., JAMA, 281(5):427-431, 1999). Antibiotics such as quinolones or quinones could reduce onset rate of acute myocardial infarction, on the other hand, antibiotics of macrolide which are known to be the most effective agents to kill Chlamydia pneumoniae have no effect on reducing the onset rate of acute myocardial infarction, suggesting that the antibiotics are not merely killing pathogenic microorganisms. Thus, antibiotics have been regarded as thrombosis inhibitors or thrombolytic agents, however, there is no evidence of relations between antibiotics and thrombus, hence, antibiotics can be conjectured to work on acute myocardial infarction via other mechanism than involvement of thrombus. The fact that antibiotics exert a certain effect on acute myocardial infarction without involvement of thrombus implies that antibiotics may protect cells from destruction caused by inadequate supply of oxygen and glucose due to the blockage of blood vessels. Accordingly, it could be expected that the patient who has ischemia due to the blockage of blood vessel can be recovered by using antibiotics, however, there is still little progress in researches of this area.

[0006] Under the circumstances, there is a continuing need to understand the effect of antibiotics on the cells under hypoxic and hypoglycemic condition to contrive its potential application in the art.

SUMMARY OF THE INVENTION

[0007] The present inventors have made an effort to elucidate the effect of antibiotics on the cells under a low oxygen (hypoxic) and a low glucose (hypoglycemic) condition, and, based on the fact that the death of cells under hypoxic and hypoglycemic condition is progressed via apoptosis, they discovered that the addition of antibiotics of quinolones, quinones, aminoglycosides or chloramphenicol to the cells under hypoxic and hypoglycemic condition can dramatically inhibit apoptosis, furthermore, the apoptosis can be inhibited by administering the antibiotics to an individual under ischemic condition in an amount of administering to the individual infected with pathogenic microorganisms.

[0008] A primary object of the present invention is, therefore, to provide a method for inhibiting apoptosis under ischemic condition which lacks an adequate supply of oxygen and glucose.

BRIEF DESCRIDTION OF THE DRAWINGS

[0009] The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:

[0010]FIG. 1 is a graph showing HepG2 cell viability under various oxygen conditions.

[0011]FIG. 2a is a graph showing the dependency of HepG2 cell viability on glucose concentration with culture time under a low oxygen condition.

[0012]FIG. 2b is a graph showing the change in residual glucose concentration with time depending on initial glucose concentration under a low oxygen condition.

[0013]FIG. 2c a graph showing the change in pH with time depending on glucose concentration under a low oxygen condition.

[0014]FIG. 3 is a graph showing HepG2 cell viability at various geneticin concentrations.

[0015]FIG. 4a is a graph showing HepG2 cell viability with culture time under a low oxygen and a low glucose condition.

[0016]FIG. 4b is a graph showing the change in glucose concentration with time under a low oxygen and a low glucose condition.

[0017]FIG. 4c is a graph showing the change in pH with time under a low oxygen and a low glucose condition.

[0018]FIG. 5a is a graph showing the HepG2 cell viability with time under a low oxygen and a high glucose condition.

[0019]FIG. 5b is a graph showing the change in glucose concentration with time under a low oxygen and a high glucose condition.

[0020]FIG. 5c is a graph showing the change in pH with time under a low oxygen and a high glucose condition.

[0021]FIG. 6a is a graph showing HepG2 cell viability with culture time under a normal oxygen and a low glucose condition.

[0022]FIG. 6b is a graph showing the change in glucose concentration with time under a normal oxygen and a low glucose condition.

[0023]FIG. 6c is a graph showing the change in lactic acid concentration with time under a normal oxygen and a low glucose condition.

[0024]FIG. 7 is a photograph showing gel electrophoresis pattern of DNA from cells treated with various antibiotics.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present inventors, first of all, examined that apoptosis is induced in cells under ischemic condition which lacks an adequate flow of blood to supply oxygen and glucose due to blockage of blood vessels by thrombus or other causes, and acknowledged that glucose generates energy via TCA cycle and electron transfer system in the presence of oxygen, however, under hypoxic (low oxygen) condition, glucose is converted to lactic acid which generates only a little energy, and resumes energy generation if sufficient oxygen is supplied.

[0026] In order to simulate ischemic cells which lack the supply of oxygen and glucose due to the blockage of blood vessels by thrombus or other causes, the present inventors created ischemic condition by discontinuing supply of oxygen and glucose to the cultured cells, and then observed the changes occurred in the cells. When oxygen was depleted in the cells, glucose also became depleted and the cells were died without utilization of lactic acid. When oxygen supply was resumed immediately after depletion of glucose, cells could survive until lactic acid was used up, that is, cells died with exhaustion of lactic acid. However, when cells were treated with antibiotics of quinolones, quinones, aminoglycosides or chloramphenicol, it was found that the cells were viable for a certain period of time even after exhaustion of glucose and lactic acid. Preferably, the antibiotics include, but are not intended to be limited to, quinolones of 10-100 μg/ml levofloxacin, 10-100 μg/ml ofloxacin or 1-10 μg/ml ciprofloxacin; quinones of tetracycline, minocycline, doxycycline, or oxytetracycline at a concentration of 0.1-10 μg/ml each; and, aminoglycosides of 10-100 μg/ml geneticin, 500-1000 μg/ml neomycin or 100-1000 μg/ml gentamycin. In case of chloramphenicol, a concentration of 1-10 μg/ml were preferably employed.

[0027] Analyses of various test groups of cells under a condition of oxygen and glucose depletion have shown that the groups of cells without antibiotic treatment underwent typical apoptosis, whereas, the groups of cells treated with said antibiotics did not undergo apoptosis for a certain period of time. These results imply that the said antibiotics inhibit apoptosis occurred in cells with ischemic injury which lacks an adequate supply of oxygen and glucose. Additional experiments demonstrated that antibiotics somehow affect the expression of bcl-2 protein which is known to be an inhibitor of apoptosis in cells with ischemic injury.

[0028] In order to examine if the results obtained with cultured cells in vitro can be applied to the tissue with ischemic injury, the mice under ischemic condition were treated with the said antibiotics and then hearts from the mice with or without antibiotic treatment were subject to biopsy, and found that the preservation rate of cardiac tissues from mice treated with the antibiotics was higher than that without antibiotic treatment.

[0029] The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

EXAMPLE 1 Cell Viability under Various Oxygen Conditions

[0030] HepG2 cells (human hepatoma cell line, ATCC HB 8065, 1×10⁶ cells/60 mm culture dish) were grown in a minimal essential medium supplemented with 100 unit/ml penicillin, 100 μg/ml streptomycin, 1 g/l glucose, 2.2 g/l sodium bicarbonate, and 10% (w/v) fetal calf serum for 2 days, followed by feeding with the same medium and incubating under an environment of 1, 2, or 5% (v/v) oxygen, respectively. Numbers of viable cells with time were determined by trypan blue exclusion assay using hemocytometer after 10-15 minutes of incubation of 1:1 (v/v) mixture of 0.4% (w/v) trypan blue and cell suspension. Cell viability with time was represented in the ratio of viable cell number to cell number just before the incubation condition was changed to a low oxygen condition (see: FIG. 1). FIG. 1 is a graph showing cell viability under various oxygen conditions, where (e) indicates 1% (v/v), (▾) indicates 2% (v/v), (▪) indicates 5% (v/v), and (♦) indicates 21% (v/v) oxygen, respectively. As shown in FIG. 1, it was clearly demonstrated that HepG2 cells were viable in a minimal medium containing low concentration of glucose under an environment over 5% (v/v) oxygen, whereas, the cells died under an environment of less than 2% (v/v) oxygen. Accordingly, a low oxygen condition was set at 1% (v/v) oxygen in the following examples.

EXAMPLE 2 Cell Viability Depending on Glucose Concentration

[0031] HepG2 cells were cultured analogously as in Example 1 except for 1% (v/v) oxygen and varied glucose concentrations from 1 to 4.5 g/L. Then, cell viability, changes in glucose concentration and changes in pH with time were measured (see: FIGS. 2a, 2 b and 2 c). FIG. 2a shows cell viability with culture time, 2 b shows changes in glucose concentration with time, and 2 c shows changes in pH with time, where () indicates 1 g/L glucose, (◯) indicates 2 g/L glucose, (▾) indicates 3 g/L glucose, (∇) indicates 3.5 g/L glucose, (▪) indicates 4 g/L glucose, and (□) indicates 4.5 g/L glucose, respectively. As shown in FIGS. 2a-2 c, it was clearly demonstrated that cells were died as a result of depletion of glucose or lowering of pH under a low oxygen condition.

EXAMPLE 3 Cell Viability under Various Geneticin Concentrations

[0032] HepG2 cells were cultured for 2 days in the same manner as in Example 1, and then, the maximum concentration of geneticin at which HepG2 cells can survive was determined by replacing the culture medium with a fresh medium containing 0-1000 μg/ml geneticin, an aminoglycoside antibiotic under an environment of 1% (v/v) oxygen, respectively (see: FIG. 3). FIG. 3 is a graph showing the cell viability at various geneticin concentrations, where geneticin was added at a concentration of 0 μg/ml (), 1 μg/ml (◯), 3 μg/ml (▾), 10 μg/ml (∇) , 100 μg/ml (▪) , and 1000 μg/ml (□), respectively. As shown in FIG. 3, it was clearly demonstrated that cells treated with 10-100 μg/ml genticin were viable for a certain period of time under an environment of 1% (v/v) oxygen.

EXAMPLE 4 Dependency of Cell Viability on Geneicin Concentration

[0033] Concentration dependency of cell viability on geneticin was determined under a low glucose (1 g/L) or a high glucose (4.5 g/L) condition, as well as under a low oxygen (1%, v/v) or normal oxygen condition.

EXAMPLE 4-1 Cell Viability under a Low Oxygen (Hypoxic) and a Low Glucose (Hypoglycemic) Condition

[0034] HepG2 cells were plated in 60 mm culture dishes at a density of 5×10⁵ cells per dish under a condition of 1 g/L glucose and 1% (v/v) oxygen, and then, cell viability, changes in pH and changes in glucose concentration were determined with time after adding 10 μg/ml geneticin or without addition, respectively (see: FIGS. 4a, 4 b and 4 c). FIG. 4a shows HepG2 cell viability with incubation time, 4 b shows change in glucose concentration with incubation time, and 4 c shows change in pH with incubation time, where () indicates without addition and (◯) indicates addition of 10 μg/ml geneticin. As shown in FIGS. 4a-4 c, it was clearly demonstrated that geneticin maintained cell viability even after glucose was used up under hypoxic and hypoglycemic condition.

EXAMPLE 4-2 Cell Viability under a Low Oxygen (Hypoxic) and a High Glucose Condition

[0035] HepG2 cells were grown analogously as in Example 4-1, except for 4.5 g/L glucose and 1% (v/v) oxygen, and then, cell viability, changes in pH and changes in glucose concentration were determined with time after treatment with 10 μg/ml geneticin or without treatment, respectively (see: FIGS. 5a, 5 b and 5 c). FIG. 5a shows HepG2 cell viability with incubation time, 5 b shows changes in glucose concentration with incubation time, and 5 c shows change in pH with incubation time, where () indicates without treatment and (◯) indicates treatment with 10 μg/ml geneticin. As shown in FIGS. 5a-5 c, it was clearly demonstrated that geneticin maintained cell viability under a hypoxic and high glucose condition in a similar manner like under hypoxic and hypoglycemic condition.

EXAMPLE 4-3 Cell Viability under a Normal Oxygen (Normoxic) and a Low Glucose (Hypoglycemic) Condition

[0036] HepG2 cells were grown analogously as in Example 4-1, except for 1 g/L glucose and 21% (v/v) oxygen, and then, cell viability, change in glucose concentration and change in lactic acid concentration were determined with time after adding 10 μg/ml geneticin or without addition, respectively (see: FIGS. 6a, 6 b and 6 c). FIG. 6a shows HepG2 cell viability with incubation time, 6 b shows change in glucose concentration with incubation time, and 6 c shows change in lactic acid concentration with incubation time, where () indicates without treatment and (▪) indicates treatment with 10 μg/ml geneticin. As shown in FIGS. 6a-6 c, it was clearly demonstrated that under normoxic condition, cells survived while consuming accumulated lactic acid even after depletion of glucose and died with exhaustion of lactic acid, whereas, cells treated with geneticin were viable without being affected by depletion of lactic acid.

EXAMPLE 5 Screening of Antibiotics Exerting Effects on Cell Viability

[0037] In order to examine whether antibiotics with other structures than aminoglycoside antibiotic of geneticin, can also enhance cell viability under a hypoxic condition, analyses were performed as followings: i.e., after analysis of antibiotics such as geneticin, neomycin, gentamycin, tetracycline, minocycline, oxytetracycline, doxycycline, chloramphenicol, levofloxacin, ofloxacin, ciprofloxacin, ampicillin, amoxicillin, cephalosporin, erythromycin, sulfadiazine, cyclohexamide, 5-fluorouracil, puromycin and trimetazidine in accordance with the procedure described in Examples 4-1 and 4-2, antibiotics which showed enhancement of cell viability under hypoxic condition were selected and their effective concentrations were determined, respectively (see: Table 1). TABLE 1 Antibiotics exerting enhancement effects on cell viability and their effective concentration Antibiotics Concentration (μg/ml) geneticin 10-100 neomycin 1000 gentamicin 100-1000 tetracycline 0.1-10   minocycline 0.1-10   doxycycline 0.1-10   oxytetracycline 0.1-10   chloramphenicol 1-10 levofloxacin 10-100 ofloxacin 10-100 ciprofloxacin 1-10

[0038] Effective concentration ranges in Table 1 represent the concentration ranges of antibiotics exerting enhancement effects on HepG2 cell viability under 1% (v/v) oxygen condition. As shown in Table 1 above, among the antibiotics known to act on 30S subunit of ribosome in E. coli, neomycin and gentamycin other than geneticin were effective among aminoglycoside antibiotics. Also, among the antibiotics known to act on 30S subunit of ribosome in E. coli, a quinone antibiotic of tetracycline was effective at very low concentration range of 0.1-10 μg/ml and tetracycline derivatives such as minocycline, oxytetracycline and doxycycline were effective at the same range of low concentration. Meanwhile, among the antibiotics known to act on 50S subunit of ribosome in E. coli, an aromatic antibiotic of chloramphenicol was effective, but a macrolide antibiotic of erythromycin was not effective. Among quinolone antibiotics known to act on DNA gyrase, all analyzed compounds, levofloxacin, ofloxacin, and ciprofloxacin were effective. However, antibiotics known to inhibit synthesis of cell wall of microorganisms, such as ampicillin, amoxillin, and cephalosporin did not show enhancement effect on cell viability. Antibiotics such as a sulfadiazine which is known to inhibit dihydropteroate synthetase in the folic acid metabolism, a cyclohexamide inhibiting protein synthesis in eukaryotes, a 5-fluorouracil blocking DNA synthesis by competing with uracil, and puromycin inhibiting protein synthesis did not show any effect on cell viability. Based on these results, it has been demonstrated that there is no significant relations between the ability of antibiotics to enhance cell viability under hypoxic condition and the action mechanism of antibiotics or the chemical structure of antibiotics. Although efficacy of antibiotics to maintain cell viability under hypoxic condition varies, effective concentration range of antibiotics on enhancement of viability of human hepatoma cell line was about 0.1 to 1000 μg/ml. Meanwhile, trimetazidine which is known to enhance cell viability by increasing utilization of glucose under a hypoxic condition did not show any positive result in the experiment.

EXAMPLE 6 Analysis of DNA

[0039] Since it has been demonstrated that antibiotics of quinolones, quinones, and aminoglycosides enhanced cell viability under a hypoxic condition in Example 5, DNA samples extracted from the cells treated with various kind of antibiotics were analyzed and compared with DNA samples from cells without antibiotic treatment.

[0040] HepG2(Human hepatoma cell line, ATCC HB 8065) cells were grown under the same condition described in Example 1 for 2 days, and then, the medium was replaced with a medium proper for test conditions described below, followed by incubating for 2 days: test group A with 21% (v/v) oxygen and 4.5 g/L glucose, test group B with 21% (v/v) oxygen and 1 g/L glucose, test group C with 1% (v/v) oxygen and 1 g/L glucose, test group D is an aminoglycoside antibiotic of geneticin (10 μg/ml) treated test group C, test group E is a quinolone antibiotic of ofloxacin (10 μg/ml treated test group C, test group F is a quinone antibiotic of doxycycline (0.1 μg/ml treated test group C. DNA samples exracted from the cells of said test groups were subject to electrophoresis, respectively, to compare DNA patterns (see: FIG. 7). FIG. 7 is a photograph showing gel electrophoresis pattern of DNA from cells of various test groups, where lane 1 indicates a size marker, lane 2, test group A, lane 3, test group B, lane 4, test group C, lane 5, test group D, lane 6, test group E, and, lane 7, test group F, respectively. As shown in FIG. 7, laddering was observed in test group C, on the other hand, almost no laddering was observed in test groups D, E and F, suggesting that, under hypoxic and hypoglycemic condition, cell death is progressed via apoptosis, but, apoptic cell death can be inhibited by treatment with antibiotics.

[0041] As clearly illustrated and demonstrated above, the present invention provides a method for inhibiting apoptosis under ischemic condition. The method for inhibiting apoptosis under ischemic condition comprises a step of administering antibiotics of quinolones, quinones, aminoglycosides or chloramphenicol to an individual under ischemic condition which lacks an adequate supply of oxygen and glucose. In accordance with the present invention, the antibiotics increase cell viability under hypoxic and hypoglycemic condition, assuring that they can be applied as a therapeutic agent for ischemia-associated diseases such as myocardial infarction and cerebral infarction. 

What is claimed is:
 1. A method for inhibiting apoptosis under ischemic condition, which comprises a step of administering antibiotics to an individual under ischemic condition in an amount of administering to the individual infected with pathogenic microorganisms.
 2. The method for inhibiting apoptosis under ischemic condition of claim 1, wherein the antibiotics are quinolones, quinones and aminoglycosides.
 3. The method for inhibiting apoptosis under ischemic condition of claim 2, wherein the quinolone antibiotics are levofloxacin, ofloxacin and ciprofloxacin.
 4. The method for inhibiting apoptosis under ischemic condition of claim 2, wherein the quinone antibiotics are tetracycline, minocycline, doxycycline and oxycycline.
 5. The method for inhibiting apoptosis under ischemic condition of claim 2, wherein the aminoglycoside antibiotics are geneticin, neomycin and gentamycin. 